Evaluation_of_Herbal_Drugs.pptx

EVALUATION OF HERBAL DRUGS
NATURAL AND PHARMACEUTICS
Mohanad AlBayati
Mohanad AbdulSattar Ali Al-Bayati, BVM&S, MSc. Physiol., PhD.
Assistant Professor of Pharmacology and Toxicology
Department of Physiology and Pharmacology
College of Veterinary Medicine
University of Baghdad
Al Ameria, Baghdad
Phone: 0964 7700766650
E. Mail: aumnmumu@covm.uobaghdad.edu.iq
aumnmumu@yahoo.com

INTRODUCTORY
Traditional herbal medicine and their preparations
have been widely used for the thousands of years in
developing and developed countries owing to its natural
origin and lesser side effects or dissatisfaction with the
results of synthetic drugs.
This may be the main reason why quality control of
oriental herbal drugs is more difficult than that of synthetic
drug. As pointed in “General Guidelines for Methodologies
on Research and Evaluation of Traditional Medicines.
(World Health Organization, 2000)

The chemistry of plants involves the presence of
therapeutically important constituents usually associated
with many inert substances (coloring agents, cellulose,
lignin etc). The active principles are extracted from the
plants and purified for therapeutic utility for their selective
pharmacological activity.

HERBAL PHARMACEUTICS SHORT COMES
Drug adulteration
With artificially manufactured material
Substances artificially manufactured being resemble with
original drug are used as substitutes. This practice is generally
followed for much costlier drug e.g. nutmeg is adulterated with
basswood prepared to the required shape and size, the colored
paraffin wax is used in place of beeswax.

With inferior quality materials
Inferior quality material may or may not have same chemical or
therapeutic value as that of original natural drug due to their
morphological resemblance to authentic drug, they are
marketed as adulterants e.g. Belladonna leaves are
substituted with Ailanthus leaves, papaya seeds to adulterate
Piper nigrum, mother cloves and clove stalks are mixed with
clove, beeswax is substituted by Japan wax.

With exhausted material
Many drugs extracted on large scale for isolation of active
principle, volatile oils etc. the exhausted material may be
used entirely or in part as a substituent for the genuine
drug e.g. umbelliferous fruits and cloves (without volatile
oils) are adulterated with exhausted (without volatile oils)
original drugs, exhausted jalap and Indian hemp (without
resins) are used as adulterant.

With foreign matter
Sometimes synthetic chemicals are used to enhance the
natural character e.g. addition of benzyl benzoate to
balsam of Peru, citral to citrus oils like oil of lemon and
orange oil etc.

With harmful / Fictitious substances
Sometimes the wastes from market are collected and
admixed with authentic drugs particularly for liquids or
unorganized drugs e.g. pieces of amber colored glass in
colophony, limestone in asafetida, lead shot in opium, white
oil in coconut oil, cocoa butter with stearin or paraffin.

Faulty collection
Some of the herbal adulteration is due to the carelessness
of herbal collectors and suppliers. The correct part of
genuine plant should be collected. Other less valuable part
of the genuine plant should not be collected. Moreover
collection should be carried out at a proper season and
time when the active constituents reach maximum. Datura
strumarium leaves should be collected during flowering
stage and wild cherry bark in autumn etc.

Imperfect preparation
Non removal of associated structures eg stems are
collected with leaves, flowers, fruits. Non-removal of
undesirable parts or structures e.g. cork should be
removed from ginger rhizome. Proper drying conditions
should be adhered. Improper drying may lead to
unintentional adulteration e.g. if digitalis leaves are dried
above 65oc decomposition of glycosides by enzymatic
hydrolysis. Use of excessive heat in separating the code
liver oil from livers, where the proportions of vitamins, odor
and color etc are adversely affected.

Incorrect storage
Deterioration especially during storage, leading to the loss of the active
ingredients, production of metabolites with no activity and, in extreme
cases, the production of toxic metabolites. Physical factors such as air
(oxygen), humidity, light, and temperature can bring about deterioration
directly or indirectly. These factors, alone or in combination, can lead to
the development of organisms such as molds, mites, and bacteria.
Oxidation of the constituents of a drug can be brought about by oxygen
in the air, causing some products, such as essential oils, to resinify or
to become rancid. Moisture or humidity and elevated temperatures can
accelerate enzymatic activities, leading to changes in the physical
appearance and decomposition of the herb. For example volatile oils
should be protected from light and stored in well closed containers in
cool place. Belladonna leaf should be stored in moisture free
containers, which may cause enzymatic action lead to decomposition of
medicinally active constituents. Mites, nematode worms, insects/moths,
and beetles can also destroy herbal drugs during storage.

Gross substitution with plant material
Due to morphological resemblance i.e similarity in
appearance, colors etc the genuine crude drugs are
substituted with others are very often sold in the market
e.g. Podophyllum peltatum L. is used as a substitute for P.
hexandrum, Belladona leaves are substituted with
Ailanthus leaves, saffron is admixed with dried flowers of
Carthamus tinctorius, mother cloves and clove stalks are
mixed with clove.

Substitution with exhausted drugs
In this type, the same drug is admixed but devoid of any
medicinally active constituents as they are already
extracted out. This practice is more common in case of
volatile oil containing drugs like fennel, clove, coriander,
caraway etc. sometime, natural characters of exhausted
drugs like color and taste are manipulated by adding other
additives and then it is substituted eg exhausted gentian
made bitter with aloes.

PRINCIPLES OF HERBAL PHARMACEUTICS
Chemical evaluation
Most of drugs have definite chemical constituents to which
their biological or pharmacological activity is attributed.
Qualitative chemical test are used to identify certain drug or
to test their purity. The isolation, purification, identification
of active constituents is based on chemical methods of
evaluation. Qualitative chemical test such as acid value,
saponification value etc. Some of these are useful in
evaluation of resins (acid value, sulphated ash), balsams
(acid value, saponification value and bester values), volatile
oils (acetyl and ester values) and gums (methoxy
determination and volatile acidity). Preliminary
phytochemical screening is a part of chemical evaluation.
These qualitative chemical tests are useful in identification
of chemical constituents and detection of adulteration.

Physical evaluation
Physical constants are sometimes taken into consideration
to evaluate certain drugs. These include moisture content,
specific gravity, optical rotation, refractive, melting point,
viscosity and solubility in different solvents. All these
physical properties are useful in identification and detection
of constituents present in plant.

Storage
Medicinal plant materials must be stored under specified
conditions in order to avoid contamination and
deterioration.
Containers
The container and its closure must not interact physically or
chemically with the material within in any way that would
alter its quality.

Protection from light
Medicinal plant materials requiring protection from light
should be kept in a light-resistant container that - either by
reason of the inherent properties of the material from which
it is made or because a special coating has been applied to
it - shields the contents from the effects of light.
Alternatively, the container may be placed inside a suitable
light-resistant (opaque) covering and/or stored in a dark
place.

Temperature
Materials that need to be stored at temperatures other than
room temperature should be labelled accordingly.
Humidity
Low humidity may be maintained, if necessary, by the use
of a desiccant in the container provided that direct contact
with the product is avoided. Care must be taken when the
container is opened in damp or humid conditions.

Size of cut
Medicinal plant materials are used either whole, or in cut or
powdered form.
Cut medicinal plant materials are prepared by cutting or
crushing the plant into small pieces. The cut is graded
according to the aperture size of the mesh of the sieve
through which the material will pass, and is indicated as
follows:
Aperture size (mm)
coarse cut 4.00
medium cut 2.80
fine cut 2.00

Powders
The coarseness or fineness of a powder is classed
according to the nominal aperture size expressed in hum of
the mesh of the sieve through which the powder will pass,
and is indicated as follows:

Sampling of material in bulk
If initial inspection indicates that the batch is uniform, take
samples as follows. When a batch consists of five
containers or packaging units, take a sample from each
one. From a batch of 6-50 units, take a sample from five. In
the case of batches of over 50 units, sample 10%, rounding
up the number of units to the nearest multiple of ten. For
example, a batch of 51 units would be sampled as for 60,
i.e. take samples from six packages.

After opening, inspect the contents of the units selected for
sampling for:
- organoleptic characteristics (colour, texture and odour);
- presentation of the material (raw, cut, crushed,
compressed);
- the presence of admixtures, foreign matter (sand, glass
particles, dirt), mould, or signs of decay;
- the presence of insects;
- the presence of packaging material originating from poor
or degraded containers.
pooled sample
average sample
final samples
degree of fragmentation (sieve test);
- identity and level of impurities;
- moisture and ash content;
- level of active ingredients, where possible.

Determination of foreign matter
Medicinal plant materials should be entirely free from visible signs of
contamination by moulds or insects, and other animal contamination,
including animal excreta. No abnormal odour, discoloration, slime or
signs of deterioration should be detected. It is seldom possible to obtain
marketed plant materials that are entirely free from some form of
innocuous foreign matter. However, no poisonous, dangerous or
otherwise harmful foreign matter or residue should be allowed.
During storage, products should be kept in a clean and hygienic place,
so that no contamination occurs. Special care should be taken to avoid
formation of moulds, since they may produce aflatoxins.
Macroscopic examination can conveniently be employed for determining
the presence of foreign matter in whole or cut plant materials. However,
microscopy is indispensable for powdered materials.
Any soil, stones, sand, dust and other foreign inorganic matter must be
removed before medicinal plant materials are cut or ground for testing.

Foreign matter is material consisting of any or all of the
following:
- parts of the medicinal plant material or materials other
than those named with the limits specified for the plant
material concerned;
- any organism, part or product of an organism, other than
that named in the specification and description of the plant
material concerned;
- mineral admixtures not adhering to the medicinal plant
materials, such as soil, stones, sand, and dust.

Visual examination and odour Wrinkled and contracted
leaves, herbs or flowers should be softened and stretched
flat ("Preliminary treatment"). Certain fruits and seeds may
also require softening before dissection and observation of
internal characteristics. No other preliminary treatment is
required.

Size
A graduated ruler in millimetres is adequate for the
measurement of the length, width and thickness of crude
materials. Small seeds and fruits may be measured by
aligning 10 of them on a sheet of calibrated paper, with 1
mm spacing between lines, and dividing the result by 10.
Colour
Examine the untreated sample under diffuse daylight. If
necessary, an artificial light source with wavelengths similar
to those of daylight may be used. The colour of the sample
should be compared with that of a reference sample.

Surface characteristics, texture and fracture characteristics
Examine the untreated sample. If necessary, a magnifying
lens (6x to 10x) may be used. Wetting with water or
reagents, as required, may be necessary to observe the
characteristics of a cut surface. Touch the material to
determine if it is soft or hard; bend and rupture it to obtain
information on brittleness and the appearance of the
fracture plane - whether it is fibrous, smooth, rough,
granular, etc.

Odour
If the material is expected to be innocuous, place a small
portion of the sample in the palm of the hand or a beaker
of suitable size, and slowly and repeatedly inhale the air
over the material. If no distinct odour is perceptible, crush
the sample between the thumb and index finger or
between the palms of the hands using gentle pressure. If
the material is known to be dangerous, crush by
mechanical means and then pour a small quantity of
boiling water onto the crushed sample in a beaker. First,
determine the strength of the odour (none, weak, distinct,
strong) and then the odour sensation (aromatic, fruity,
musty, mouldy, rancid, etc.). A direct comparison of the
odour with a defined substance is advisable (e.g.
peppermint should have an odour similar to menthol,
cloves an odour similar to eugenol).

Clarification of microscopic particles
The presence of certain cell contents, such as starch
grains, aleurone grains, plastids, fats and oils, may render
sections non-translucent and obscure certain
characteristics. Reagents that dissolve some of these
contents can be used in order to make the remaining parts
stand out clearly or produce a penetrating effect. This
renders the section more transparent and reveals details of
the structures.
If the refractive index of the clarifying agent is close to that
of the cell structure, the material being tested becomes
almost invisible; if it differs appreciably, the material
becomes markedly evident.

Chloral hydrate TS
On gentle heating chloral hydrate TS dissolves starch
grains, aleurone grains, plastids, and volatile oils, and
expands collapsed and delicate tissue without causing any
undue swelling of cell walls or distortion of the tissues. It
has a refractive index (n) of 1.44 -1.48. It is the best
reagent for rendering calcium oxalate clearly evident and is
particularly useful for small crystals. However, when
allowed to stand, it slowly dissolves calcium oxalate, owing
to an increase in acidity.

Lactochloral TS
Lactochloral TS has a similar use to chloral hydrate TS, but
is usually applied to sections that are difficult to clarify. It
may be used cold. Before use, any air present in the
specimen should be removed by placing in a desiccator
and applying a vacuum.

Lactophenol TS
Lactophenol TS may be used cold or with heating. It has a
refractive index () of 1.44 and is useful for the preparation
of fungi, pollen grains, most non-oily powders, and
parasites such as mites and nematode worms. Sizes of
starch grains can be measured accurately, but the
concentric rings are usually invisible when prepared in this
reagent. Crystals of calcium oxalate are clearly visible in
lactophenol and shine brightly when illuminated with
polarized light. This reagent dissolves calcium carbonate
deposits with a slow effervescence, owing to the presence
of lactic acid.

Sodium hypochlorite TS
Sodium hypochlorite TS is used for bleaching deeply
coloured sections. Immerse the sections in the solution for a
few minutes until sufficiently bleached, wash with water and
prepare the mount with glycerol/ethanol TS. The bleached
sections give a negative reaction to lignin.

Solvents for fats and oils
Xylene R and light petroleum R can be used to remove fats
and oils from oily powders or sections. When necessary,
immerse the material in the solvent for a short time, decant
the liquid and wash the material with fresh solvent.

HISTOCHEMICAL DETECTION OF CELL WALLS
AND CONTENTS
Reagents can be applied to a powdered sample or a
section on a slide by the following methods:
• Add drops of the reagent to the sample and apply a
cover-glass, then irrigate using a strip of filter-paper as
described below.
• Place drops of the reagent on one edge of the cover-
glass of a prepared specimen. Place a strip of filter-paper
at the opposite edge of the cover-glass to remove the
fluid under the cover-glass by suction, causing the
reagent to flow over the specimen.

Cellulose cell walls
Add 1-2 drops of iodinated zinc chloride TS and allow to
stand for a few minutes; alternatively, add 1 drop of iodine
(0.1 mol/l) VS, allow to stand for 1 minute, remove excess
reagent with a strip of filter-paper and add 1 drop of sulfuric
acid (∼1160g/l) TS; cellulose cell walls are stained blue to
blue-violet. On the addition of 1-2 drops of cuoxam TS, the
cellulose cell walls will swell and gradually dissolve.

Lignified cell walls
Moisten the powder or section on a slide with a small
volume of phloroglucinol TS and allow to stand for about 2
minutes or until almost dry. Add 1 drop of hydrochloric acid
(∼420 g/l) TS and apply a cover-glass; lignified cell walls
are stained pink to cherry red.

Suberized or cuticular cell walls
Add 1-2 drops of sudan red TS and allow to stand for a few
minutes or warm gently; suberized or cuticular cell walls
are stained orange-red or red.

Add a few drops of iodine/ethanol TS; the aleurone grains
will turn yellowish brown to brown. Then add a few drops of
ethanolic trinitrophenol TS; the grains will turn yellow. Add
about 1 ml of mercuric nitrate TS and allow to dissolve; the
colour of the solution turns brick red. If the specimen is oily,
render it fat-free by immersing and washing it in an
appropriate solvent before carrying out the test.

Calcium carbonate
Crystals or deposits of calcium carbonate dissolve slowly
with effervescence when acetic acid (∼60g/l) TS or
hydrochloric acid (∼70g/l) TS is added.

Calcium oxalate
Crystals of calcium oxalate are insoluble in acetic acid
(∼60g/l) TS but dissolve in hydrochloric acid (∼70g/l) TS
without effervescence (if applied by irrigation the acid
should be more concentrated); they also dissolve in sulfuric
acid (∼350g/l) TS, but needle-shaped crystals of calcium
sulfate separate on standing after about 10 minutes. In
polarized light, calcium oxalate crystals are birefringent.
Calcium oxalate is best viewed after the sample has been
clarified, e.g. with chloral hydrate TS.

Fats, fatty oils, volatile oils and resins
Add 1-2 drops of sudan red TS and allow to stand for a few
minutes or heat gently, if necessary. The fatty substances
are stained orange-red to red. Irrigate the preparation with
ethanol (∼750 g/l) TS and heat gently; the volatile oils and
resins dissolve in the solvent, while fats and fatty oils
(except castor oil and croton oil) remain intact.

Hydroxyanthraquinones
Add 1 drop of potassium hydroxide (∼55 g/l) TS; cells
containing 1,8-di-hydroxyanthraquinones are stained red.

Inulin
Add 1 drop each of 1-naphthol TS and sulfuric acid
(∼1760g/l) TS; spherical aggregations of crystals of inulin
turn brownish red and dissolve.

Mucilage
Add 1 drop of Chinese ink TS to the dry sample; the
mucilage shows up as transparent, spherically dilated
fragments on a black background. Alternatively, add 1 drop
of thionine TS to the dry sample, allow to stand for about 15
minutes, then wash with ethanol (∼188g/l) TS; the mucilage
turns violet-red (cellulose and lignified cell walls are stained
blue and bluish violet respectively).

Starch
Add a small volume of iodine (0.02 mol/l) VS; a blue or
reddish blue colour is produced. Alternatively, add a small
volume of glycerol/ethanol TS and examine under a
microscope with polarized light; birefringence is observed
giving a Maltese cross effect with the arms of the cross
intersecting at the hilum.

Tannin
Add 1 drop of ferric chloride (50g/l) TS; it turns bluish black
or greenish black.

Disintegration of tissues
Cut the material into small pieces, about 2 mm thick and 5
mm long or into slices, about 1 mm thick (tangential
longitudinal sections are preferred for woods or xylem from
stems).
Use one of the following methods depending on the nature
of the cell walls. For tissues with lignified cell walls use
either method 1 or 2. For tissues where lignified cells are
few or occur in small groups, use method 3.

Method 1. Nitric acid and potassium chlorate
Place the material in a test-tube containing about 5 ml of
nitric acid (∼500g/l) TS and heat to boiling. Add a small
quantity of powdered potassium chlorate R and allow to
react, warming gently if necessary to maintain a slight
effervescence; add fresh quantities of powdered
potassium chlorate R as needed. When the tissue
appears to be almost coampletely bleached and shows a
tendency to disintegrate, apply pressure with a glass rod
to the material. If the material breaks, interrupt the
reaction by pouring the contents of the test-tube into
water. Allow the material to settle, decant it and wash it
with fresh water until the acidity is removed. Transfer the
material onto a slide and tease it out with a needle. Add 1
drop of glycerol/ethanol TS and apply a cover-glass. The
disintegrated material gives a negative reaction for lignin.

Method 2. Nitric acid and chromic acid
Place the material in a small dish and heat with nitro-
chromic acid TS until the material breaks easily when
pressure is applied with a glass rod. Wash the material
repeatedly with water and transfer onto a slide. Tease out
the material, add 1 drop of glycerol/ethanol TS and apply a
cover-glass. The disintegrated material gives a negative
reaction for lignin.
This treatment can also be carried out on a slide. Place a
moderately thick section of the material on a slide, add the
reagent and allow to stand for about 20 minutes. Separate
the cells by applying gentle pressure, or with a sliding
movement of the cover-glass. This process is especially
useful when the disintegration of the tissues of a section
under the microscope needs to be observed to ascertain
where isolated cells come from.

Method 3. Caustic alkali method
Place the material in a test-tube containing about 5 ml of
potassium hydroxide (∼110g/l) TS or sodium hydroxide
(∼80 g/l) TS, and heat on a water-bath for 15-30 minutes
until a portion breaks easily when pressure is applied with a
glass
rod. Decant the liquid and wash the softened material
several times with fresh quantities of water. This method is
particularly useful for the disintegration of bark, seeds,
leaves and flowers, facilitating the elimination of fibres,
scleroids, lactiferous tissues and epidermis. The
disintegrated material gives a negative reaction for lignin.

Determination of the stomatal index
Place fragments of leaves, about 5 x 5 mm in size, in a test-tube containing about 5
ml of chloral hydrate TS and heat on a water-bath for about 15 minutes or until
the fragments are transparent. Transfer a fragment to a slide and prepare it as
described earlier, the lower epidermis uppermost, in chloral hydrate TS; place a
small drop of glycerol/ethanol TS at one side of the cover-glass to prevent the
material from drying. Examine under a microscope with a 40x objective and a 6x
eyepiece, equipped with a drawing apparatus. Mark on the drawing paper a cross
(χ) for each epidermal cell and a circle (o) for each stoma. Calculate the stomatal
index as follows:
where S = the number of stomata in a given area of leaf
E= the number of epidermal cells (including trichomes) in the same area of leaf.
For each leaf sample, no fewer than ten determinations should be carried out and
the average index calculated.

Determination of ash
The total ash method is designed to measure the total
amount of material remaining after ignition. This includes
both "physiological ash", which is derived from the plant
tissue itself, and "non-physiological" ash, which is the
residue of the extraneous matter (e.g. sand and soil)
adhering to the plant surface.
Acid-insoluble ash is the residue obtained after boiling the
total ash with dilute hydrochloric acid, and igniting the
remaining insoluble matter. This measures the amount of
silica present, especially as sand and siliceous earth.
Water-soluble ash is the difference in weight between the
total ash and the residue after treatment of the total ash
with water.

Determination of extractable matter
Method 1. Hot extraction
Place about 4.0g of coarsely powdered air-dried material,
accurately weighed, in a glass-stoppered conical flask. Add
100ml of water and weigh to obtain the total weight including
the flask. Shake well and allow to stand for 1 hour. Attach a
reflux condenser to the flask and boil gently for 1 hour; cool
and weigh. Readjust to the original total weight with the
solvent specified in the test procedure for the plant material
concerned. Shake well and filter rapidly through a dry filter.
Transfer 25 ml of the filtrate to a tared flat-bottomed dish and
evaporate to dryness on a water-bath. Dry at 105°C for 6
hours, cool in a desiccator for 30 minutes, then weigh
without delay. Calculate the content of extractable matter in
mg per g of air-dried material.

Method 2. Cold maceration
Place about 4.0g of coarsely powdered air-dried material,
accurately weighed, in a glass-stoppered conical flask.
Macerate with 100ml of the solvent specified for the plant
material concerned for 6 hours, shaking frequently, then
allow to stand for 18 hours. Filter rapidly taking care not to
lose any solvent, transfer 25 ml of the filtrate to a tared flat-
bottomed dish and evaporate to dryness on a water-bath.
Dry at 105°C for 6 hours, cool in a desiccator for 30 minutes
and weigh without delay. Calculate the content of extractable
matter in mg per g of air-dried material.
For ethanol-soluble extractable matter, use the
concentration of solvent specified in the test procedure for
the plant material concerned; for water-soluble extractable
matter, use water as the solvent. Use other solvents as
specified in the test procedure.

Determination of water and volatile matter
An excess of water in medicinal plant materials will
encourage microbial growth, the presence of fungi or
insects, and deterioration following hydrolysis. Limits for
water content should therefore be set for every given plant
material. This is especially important for materials that
absorb moisture easily or deteriorate quickly in the
presence of water.

The azeotropic method gives a direct measurement of the
water present in the material being examined. When the
sample is distilled together with an immiscible solvent, such
as toluene R or xylene R, the water present in the sample
is absorbed by the solvent. The water and the solvent are
distilled together and separated in the receiving tube on
cooling. If the solvent is anhydrous, water may remain
absorbed in it leading to false results. It is therefore
advisable to saturate the solvent with water before use.

Apparatus used to determine
water content by the
azeotropic method
(dimensions in mm)
The apparatus consists of a glass
flask (A) connected by a tube (D) to
a cylindrical tube (B) fitted with a
graduated receiving tube (E) and a
reflux condenser (C). The receiving
tube (E) is graduated in 0.1-ml
divisions so that the error of
readings does not exceed 0.05 ml.
The preferred source of heat is an
electric heater with a rheostat
control, or an oil-bath. The upper
portion of the flask and the
connecting tube may be insulated.

The test for loss on drying determines both water and
volatile matter. Drying can be carried out either by heating
to 100-105 °C or in a desiccator over phosphorus
pentoxide R under atmospheric or reduced pressure at
room temperature for a specified period of time. The
desiccation method is especially useful for materials that
melt to a sticky mass at elevated temperatures.

Loss on drying (gravimetric determination)
Place about 2-5g of the prepared air-dried material, or the
quantity specified in the test procedure for the plant material
concerned, accurately weighed, in a previously dried and
tared flat weighing bottle. Dry the sample by one of the
following techniques:
- in an oven at 100-105°C;
- in a desiccator over phosphorus pentoxide R under
atmospheric pressure or reduced pressure and at room
temperature.
Dry until two consecutive weighings do not differ by more
than 5mg, unless otherwise specified in the test procedure.
Calculate the loss of weight in mg per g of air-dried material.

Determination of volatile oils
Volatile oils are characterized by their odour, oil-like appearance and
ability to volatilize at room temperature. Chemically, they are usually
composed of mixtures of, for example, monoterpenes, sesquiterpenes
and their oxygenated derivatives. Aromatic compounds predominate in
certain volatile oils.
Because they are considered to be the "essence" of the plant material,
and are often biologically active, they are also known as "essential oils".
The term "volatile oil" is preferred because it is more specific and
describes the physical properties.
In order to determine the volume of oil, the plant material is distilled with
water and the distillate is collected in a graduated tube. The aqueous
portion separates automatically and is returned to the distillation flask. If
the volatile oils possess a mass density higher than or near to that of
water, or are difficult to separate from the aqueous phase owing to the
formation of emulsions, a solvent with a low mass density and a suitable
boiling-point may be added to the measuring tube. The dissolved
volatile oils will then float on top of the aqueous phase.

Apparatus
A suitable apparatus is made from resistant glass with a low expansion coefficient, and
has the following parts:
• a round-bottomed, short-necked flask, capacity 500 or 1000 ml, the internal diameter of
the ground-glass neck being 29 mm at the widest end;
• the following sections fused into one piece (Fig. 5):
- a vertical tube (AC), 210-260 mm long, with an external diameter of 13-15 mm;
- a bent tube (CDE), CD and DE each being 145-155 mm long, and having an external
diameter of 10 mm;
- a bulb-condenser (FG), 145-155 mm long;
- a tube (GH) 30-40 mm long, with a side-arm tube (HK), at an angle of 30-40°;
- a vented ground-glass stopper (K') and a tube (K) with an internal diameter of 10 mm,
the wide end being of ground glass;
- a pear-shaped bulb (J) with a volume of 3 ml;
- a tube with a volume of 1 ml (JL), graduated over 100-110 mm in divisions of 0.01 ml;
- a bulb-like swelling (L), with a volume of about 2 ml;
- a three-way tap (M);
- a connecting tube (BM), with an external diameter of 7-8 mm, which is fitted in the
middle with a security tube (N); the junction (B) should be 20-25 mm higher than the
uppermost graduation;
• a burner allowing fine control and fitted with a flue, or an electric heating device;
• a vertical support with a horizontal ring covered with insulating material.

Determination of distillation rate
given in the test procedure. After a further 10 minutes
record the volume of oil collected in the graduated tube and
subtract the volume of solvent (xylene) previously noted.
The difference represents the volume of volatile oils in the
sample of plant material taken. Calculate the oil content in
ml per 100g of plant material.

Determination of bitterness value
Medicinal plant materials that have a strong bitter taste ("bitters") are
employed therapeutically, mostly as appetizing agents. Their bitterness
stimulates secretions in the gastrointestinal tract, especially of gastric
juice.
Bitter substances can be determined chemically. However, since they are mostly
composed of two or more constituents with various degrees of bitterness, it is first
necessary to measure total bitterness by taste.
The bitter properties of plant material are determined by comparing the threshold
bitter concentration of an extract of the materials with that of a dilute solution of
quinine hydrochloride R. The bitterness value is expressed in units equivalent to
the bitterness of a solution containing 1g of quinine hydrochloride R in 2000 ml.
Safe drinking-water should be used as a vehicle for the extraction of plant
materials and for the mouth-wash after each tasting. Taste buds dull quickly if
distilled water is used. The hardness of water rarely has any significant influence
on bitterness.

The bitter sensation is not felt by the whole surface of the
tongue, but is limited to the middle section of the upper
surface of the tongue. A certain amount of training is
required to perform this test. A person who does not
appreciate a bitter sensation when tasting a solution of
0.058 mg of quinine hydrochloride R in 10 ml of water is not
suitable to undertake this determination.
The preparation of the stock solution of each individual plant
material (ST) should be specified in the test procedure. In
each test series, unless otherwise indicated, the
determination should start with the lowest concentration in
order to retain sufficient sensitivity of the taste buds.

All solutions and the safe drinking-water for mouthwashing should be at 20-25 °C.
Calculate the bitterness value in units per g using the following formula:
2000 x c

Determination of haemolytic activity
Many medicinal plant materials, especially those derived from the families
Caryophyllaceae, Araliaceae, Sapindaceae, Primulaceae, and
Dioscoreaceae contain saponins. The most characteristic property of
saponins is their ability to cause haemolysis: when added to a
suspension of blood, saponins produce changes in erythrocyte
membranes, causing haemoglobin to diffuse into the surrounding
medium.
The haemolytic activity of plant materials, or a preparation containing
saponins, is determined by comparison with that of a reference material,
saponin R, which has a haemolytic activity of 1000 units per g. A
suspension of erythrocytes is mixed with equal volumes of a serial dilution
of the plant material extract. The lowest concentration to effect complete
haemolysis is determined after allowing the mixtures to stand for a given
period of time. A similar test is carried out simultaneously with saponin R.

procedure
To prepare the erythrocyte suspension fill a glass-stoppered
flask to one-tenth of its volume with sodium citrate (36.5 g/l) TS,
swirling to ensure that the inside of the flask is thoroughly
moistened. Introduce a sufficient volume of blood freshly
collected from a healthy ox and shake immediately. Citrated
blood prepared in this way can be stored for about 8 days at 2-
4°C. Place 1 ml of citrated blood in a 50-ml volumetric flask with
phosphate buffer pH 7.4 TS and carefully dilute to volume. This
diluted blood suspension (2% solution) can be used as long as
the supernatant fluid remains clear and colourless. It must be
stored at a cool temperature.
To prepare the reference solution, transfer about 10 mg of
saponin R, accurately weighed, to a volumetric flask and add
sufficient phosphate buffer pH 7.4 TS to make 100ml. This
solution should be freshly prepared.
The extract of plant material and dilutions should be prepared as
specified in the test procedure for the plant material concerned,
using phosphate buffer pH 7.4 TS.

Determination of tannins
Tannins (or tanning substances) are substances capable of
turning animal hides into leather by binding proteins to form
water-insoluble substances that are resistant to proteolytic
enzymes. This process, when applied to living tissue, is
known as an "astringent" action and is the reason for the
therapeutic application of tannins.
Chemically, tannins are complex substances; they usually
occur as mixtures of polyphenols that are difficult to separate
and crystallize. They are easily oxidized and polymerized in
solution; if this happens they lose much of their astringent
effect and are therefore of little therapeutic value.

procedure
To prepare the plant material extract, introduce the quantity specified in the
test procedure for the plant material concerned, previously powdered to a
known fineness and weighed accurately, into a conical flask. Add 150ml of
water and heat over a boiling water-bath for 30 minutes. Cool, transfer the
mixture to a 250-ml volumetric flask and dilute to volume with water. Allow
the solid material to settle and filter the liquid through a filter-paper,
diameter 12cm, discarding the first 50ml of the filtrate.
To determine the total amount of material that is extractable into water,
evapo-rate 50.0ml of the plant material extract to dryness, dry the residue
in an oven at 105°C for 4 hours and weigh (T1).
To determine the amount of plant material not bound to hide powder that is
extractable into water, take 80.0ml of the plant material extract, add 6.0g of
hide powder R and shake well for 60 minutes. Filter and evaporate 50.0 ml
of the clear filtrate to dryness. Dry the residue in an oven at 105°C and
weigh (T2).
To determine the solubility of hide powder, take 6.0g of hide powder R, add
80.0 ml of water and shake well for 60 minutes. Filter and evaporate 50.0
ml of the clear filtrate to dryness. Dry the residue in an oven at 105°C and
weigh (T0). Calculate the quantity of tannins as a percentage using the
following formula:

Determination of swelling index
Many medicinal plant materials are of specific therapeutic or
pharmaceutical utility because of their swelling properties,
especially gums and those containing an appreciable amount
of mucilage, pectin or hemicellulose.
The swelling index is the volume in ml taken up by the swelling
of 1 g of plant material under specified conditions. Its
determination is based on the addition of water or a swelling
agent as specified in the test procedure for each individual
plant material (either whole, cut or pulverized). Using a glass-
stoppered measuring cylinder, the material is shaken
repeatedly for 1 hour and then allowed to stand for a required
period of time. The volume of the mixture (in ml) is then read.
The mixing of whole plant material with the swelling agent is
easy to achieve, but cut or pulverized material requires
vigorous shaking at specified intervals to ensure even
distribution of the material in the swelling agent.

procedure
Carry out simultaneously no fewer than three
determinations for any given material. Introduce the
specified quantity of the plant material concerned,
previously reduced to the required fineness and accurately
weighed, into a 25-ml glass-stoppered measuring cylinder.
The internal diameter of the cylinder should be about
16mm, the length of the graduated portion about 125 mm,
marked in 0.2-ml divisions from 0 to 25 ml in an upwards
direction. Unless otherwise indicated in the test procedure,
add 25 ml of water and shake the mixture thoroughly every
10 minutes for 1 hour. Allow to stand for 3 hours at room
temperature, or as specified. Measure the volume in ml
occupied by the plant material, including any sticky
mucilage. Calculate the mean value of the individual
determinations, related to 1 g of plant material.

Determination of foaming index
Many medicinal plant materials contain saponins that can
cause a persistent foam when an aqueous decoction is
shaken. The foaming ability of an aqueous decoction of
plant materials and their extracts is measured in terms of a
foaming index.

procedure
Reduce about 1 g of the plant material to a coarse powder (sieve size no.
1250), weigh accurately and transfer to a 500-ml conical flask containing
100ml of boiling water. Maintain at moderate boiling for 30 minutes. Cool and
filter into a 100-ml volumetric flask and add sufficient water through the filter
to dilute to volume.
Pour the decoction into 10 stoppered test-tubes (height 16cm, diameter
16mm) in successive portions of 1 ml, 2 ml, 3 ml, etc. up to 10 ml, and adjust
the volume of the liquid in each tube with water to 10ml. Stopper the tubes
and shake them in a lengthwise motion for 15 seconds, two shakes per
second. Allow to stand for 15 minutes and measure the height of the foam.
The results are assessed as follows.
• If the height of the foam in every tube is less than 1 cm, the foaming index
is less than 100.
• If a height of foam of 1 cm is measured in any tube, the volume of the plant
material decoction in this tube (a) is used to determine the index. If this tube
is the first or second tube in a series, prepare an intermediate dilution in a
similar manner to obtain a more precise result.
• If the height of the foam is more than 1 cm in every tube, the foaming index
is over 1000. In this case repeat the determination using a new series of
dilutions of the decoction in order to obtain a result.

Biological evaluation
Some drugs have specific biological and pharmacological
activity which is utilized for their evaluation. Actually this
activity is due to specific type of constituents present in the
plant extract. For evaluation the experiments were carried
out on both intact and isolated organs of living animals. With
the help of bioassays (testing the drugs on living animals),
strength of drug in its preparation can also be evaluated.
Some important biological evaluations are as follow:

1. Antibiotic activity
Some bacteria such as Salmonella typhi, styphylococcus
aureus and E. coli are used to determine the antiseptic
value (the degree of antiseptic activity e.g. phenol co-
efficient of certain drugs).
The activity of antibiotics is also determined by using
Klebsiella pneumonia, Micrococcus flavus, Sarcira lutea etc.
living bacteria, yeast and molds are used to evaluate certain
vitamins.
Microbiological assays by cylinder plate method and
turbidimetric method are used in evaluation.

2. Antifertility activity
Antifertility drugs include contraceptives and abortificients.
Contraceptive drugs are used to prevent pregnancy and
abortificient to terminate pregnancy. Female rats are used
for antifertility activity i.e. measure the pregnancy rate
(antiovulation and anti-implantation) and male rats are used
for antispermatogenic activity (inhibition of
spermatogenesis) and spermicidal activity (sperm motility)
of herbal drugs.

3. Hypoglycemic activity
Rabbits, rats or mice are used to test hypoglycemic activity
of plant extract. Radio-immuno assay (RIA) or Enzyme
linked immunosorbate assay (ELISA) are done for
measurement of insulin levels.

4. Neuropharmacological activity
Testing the herbal drugs with effects on central and
autonomic nervous system. CNS acting drugs like cocaine
(Erythroxylum coca), morphine (Papaver somniferum),
cannabinol (Cannabis sativa) are tested using rodents. For
testing the herbal drugs for their effects on ANS guinea pig
ileum for antispasmodic activity, rabbit jejunum for
adrenergic activity, rat phrenicnerve- diaphragm for muscle
relaxant activity, frog rectus for skeletal muscles activity.

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